We plan to investigate several important hypotheses as they relate to growing evidence that the HIV-1 co-receptor CCR5 exists in multiple conformational states and that these conformational states and their levels of expression may affect the permissiveness of HIV-1 entry. We will also examine the mechanisms of viral resistance that may develop to anti-CCR5 Mabs and CCR5 antagonists, the biologic properties of the escape viruses and how the development of resistance to this class of compounds might be inhibited. Specifically, we will use phage display techniques to isolate a large panel of human anti-CCR5 single-chain antibodies (scFvs) that are directed against a broad range of linear and conformationally sensitive CCR5 epitopes. The scFvs will be epitope mapped and conformational heterogeneity will be assessed through binding studies to different CCR5 expressing cells lines. The scFv binding affinities for CCR5, their ability to inhibit gp120 and ligand binding and the effects of CCR5 antagonists on epitope expression will also be examined. These antibodies will be used in multiparameter FACS studies to identify and determine the distribution of these conformational states in subpopulations of CEM+ T-cells, monocytes and monocyte-derived-macrophage (MDM) at rest and after stimulation, and determine if conformational states that effect the permissiveness of HIV-1 entry can be identified. Cells will also be examined from different healthy individuals to examine heterogeneity of expression patterns in the population. HIV-1 always mutates to escape from the selection pressure of any one inhibitor and another specific aim of this proposal is to evaluate the mechanisms of viral resistance to anti-CCR5 scFvs and to determine if the escape viruses are altered in their tropism and cytopathic effects. To investigate the mechanisms of neutralization resistance, suboptimal doses of purified neutralizing scFvs will be used in R5 virus challenge experiments with activated PBMCs under conditions that favor the selection of resistant viruses. Sequential virus stocks and cloned env genes from these mutant virus stocks will be prepared. Coreceptor usage will be evaluated in HIV challenge assays, fusion assays and single-round HIV entry reporter assays. Coreceptor affinity of env genes and their ability to block scFv binding will be measured by FACS binding of recombinant gp120s to CD4 and CCR5 expressing cells in the presence and absence of scFv. Escape viruses will also be tested for their ability to enter ceils with varied CCR5 density and for their direct cytopathic effects. Since combination anti-retroviral therapies have proven important in inhibiting the development of escape mutants from all classes of antiviral agents that have been examined, we will also investigate whether acquisition of resistance to small molecule CCR5 antagonists TAK-779 and AD101 and/or anti-CCR5 scFvs can be blocked by co-treatment with a neutralizing anti- CCR5 scFv, and if resistance still develops what are the biological characteristics of the double resistance viruses. We will also investigate whether an increase in binding affinity and neutralization activity of bivalent scFvlgG fusion proteins can overcome and even prevent the development of resistance. These studies are of central importance for the further clinical development of HIV-1 entry inhibitors so that we can understand how combination therapies can be developed that will prevent or slow the development of escape mutants.